11 mg/ml sodium pyruvate; GIBCO). Microorganisms Enterotoxigenic Escherichia coli (ETEC) strain 987 (O9: H-: 987P+: STa+) was kindly BIBW2992 research buy provided by Dr. M. Nakazawa, National Institute of Animal Health (Tsukuba, Japan) [19]. ETEC cells were grown in blood agar (5% sheep blood) for 24 hours at 37°C and then transferred to tryptic soy broth (TSB; Becton, Dickinson and

Company, USA) for 5 days at 37°C without shaking to get a pellicle containing piliated phase. ETEC cells were collected from the pellicle and transferred to 1L TSB and cultured 20 hours at 37°C with shaking. After incubation, the subcultures of bacteria were centrifuged at 5000 × g for 10 min at 4°C and washed with PBS (pH7.2). Finally, BMS202 ETEC cells were heat killed at 100°C for 15 minutes and then washed with PBS. Heat-stable ETEC PAMPs were suspended in DMEM for use. The following lactobacilli strains were used in this study: Lactobacillus reuteri MEP221101 and MEP221102, Lactobacillus casei MEP221103, OLL2768, MEP221104, MEP221105, MEP221106, MEP221107, MEP221108, MEP221109, MEP221114 and MEP221115, Lactobacillus rhamnosus MEP221110, MEP221111, MEP221112 and GG, Lactobacillus salivarius MEP221113, Lactobacillus jensenii TL2937 and Lactobacillus gasseri MEP221117. The lactobacilli strains were grown in de Man, Rogosa and Sharpe (MRS) medium (Difco, Detroit, USA) for 16 h at 37°C and washed with PBS (pH7.2), and heat killed

(60°C, 30 min). These bacterial samples were resuspended in DMEM, enumerated using a Petroff-Hausser counting chamber, and stored at −80°C until use [14]. Immunocytochemistry BIE cells were cultured at a cell density of 3×104 cells/well of a 12-well culture plate collagen type I-coated glass disk

(Iwaki Glass Co., Tokyo, Japan) for 3 days, (37°C, 5% CO2). BIE cells were washed with cold PBS (pH7.2) plus 2% FCS twice and then fixed with 4% paraformaldehyde/PBS solution (room temperature, 5 minutes). Following treating with PBS-T (0.2% Triton X-100) for 5 min at room temperature and washing three times with PBS. Cells were then incubated with Alexa 488 conjugated rabbit anti-TLR2 polyclonal antibody (bs-1019R-Alexa488, Resminostat Bioss Inc., Wobum, MA, USA) or Alexa 488 conjugated rabbit anti-TLR4 antibody (bs-1021R-Alexa488, Bioss) diluted 50 times with Can Get Signal solution 1 (NKB-201, TOYOBO Co., Ltd., Osaka, Japan) overnight at 4°C. Both anti-TLR2 and anti-TLR4 antibodies cross-react with bovine receptors according to Bioss Inc. datasheet. Alexa 488 conjugate rabbit IgG (20304AF488, IMGENEX, San Diego, CA, USA) was used as isotype control. Following washing three times with PBS-T and the cells were rinsed in distilled water and then mounted with FLUOROSHIELD with DAPI (AR-6501-01, ImmunoBioScience Corp, Mukilteo, WA, USA). Immunofluorescence microscopy was performed with using a AG-881 molecular weight confocal laser microscope (LSM 700, Carl Zeiss, Oberkochen, Germany).